Genetic analysis of hypothalamic retention of [3H]corticosterone in two inbred strains of mice

Brain Research, 69 ( 1974~ 77. ' • ,(': Elsevier Scientific Publishing (.ompan.~, Am~,tcrdam

77 Printed in l h e Nethcrlands

GENETIC ANALYSIS OF HYPOTHALAMIC RETENTION OF [:~H]CORTICOSTERONE IN TWO INBRED STRAINS OF MICE

B. E. E L E F T H E R I O U

The Jackson Laboratory, Bar Harbor, Me. 04609 i U.S./4.) (Accepted September 24th, 1973

SUMMARY

Determinations were made of hypothalamic retention of [l,2-aH]cortico sterone in adrenalectomized, anestrous, adult female mice of two parental strains, BALB/cBy and C57BL/6By, their reciprocal F~ hybrids, B6CF1 and CB6F1, and their 7 recombinant inbred strains, CXBD, CXBE, CXBG. CXBH, CXBI, CXBJ and CXBK, and in the backcrosses, B6CFI :/ C57BL/6By, and BCFI ~: BALB/cBy. Results indicate that the retention of corticosterone by the hypothalamus is influenced genetically by at least one locus. This finding offers unique opportunities in determining the nature and possible mechanism of control of this phenomenon by genetic analysis.

INTRODUCTION

Recent interest has been aroused in the uptake and retention by various brain regions of [aH]corticosterone6--9. Indeed, it appears that the binding of [aH]corticosterone in the brain is due exclusively to a specific corticosterone-binding protein whose properties are distinctly different from those of the serum-corticosteronebinding protein 9. Furthermore, it was demonstrated that radioactively labeled corticosterone is bound also by neuronal nuclei, and this bound hormone under in vitro conditions exchanges with unlabeled corticosterone in the medium, more slowly than either the brain-soluble-binding protein or the serum-binding protein. For the past 2 years, we have been studying the genetic determination of hypothalamic retention of [all ]corticosterone in the mouse, with the ultimate aim of arriving at a genetic model whose utilization will aid in determining possible genetic control of the mechanism of steroid binding in the brain and, specifically, in the hypothalamus. This study was conducted on specially derived inbred strains of mice known as recombinant inbred (RI) strains. They offer the advantage of arriving at genetic

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linkage and models for genetic determination of particular traits in comparatively short periods of time. RI strains are derived from the cross of two unrelated, but highly inbred progenitor strains, and then maintained independently from the F2 generation with strict inbreeding. The procedure fixes the chance recombination of genes in generations following F~, but in ever-decreasing amounts as full homozygosity is approached. The resulting battery of strains can be looked upon, in one sense, as a replicable recombinant population. The derivation and advantageous use of such strains for analyzing gene systems is described elsewhere~,:L In this paper, we report the results of a genetic analysis of hypothalamic retention of [3H]corticosterone in female mice, using the R! strains. Data indicate that this phenomenon is controlled by at least one locus. METHODS

Experiment 1

Adult female mice aged 65 days, of the RI strains CXBD, CXBE, CXBG, CXBH, CXBI, CXBJ, and CXBK, their progenitor strains BALB/cBy (C) and C57BL/6By (B6), and their reciprocal F1 hybrids B6CF1, were adrenalectomized and maintained subsequently on 0.8 ~ saline. All animals were maintained on a standard laboratory chow ad libitum. Two weeks following adrenalectomy, animals were injected intraperitoneally with [1,2-3H]corticosterone (New England Nuclear Corp., Boston, Mass. ; 50/~Ci/mmole) in 6 0 ~ ethanol-saline at a dose of 50/~Ci (0.36/~g). Routine purity checks of the hormone were carried out using chromatography, as described by others 7,s. During a pilot experiment, it was found that maximum uptake of [3H]corticosterone occurred at 40 rain after injection, followed subsequently by a rapid decline. Based on these findings, animals were killed by decapitation at 35 rain after the injection of the hormone. To avoid variation due to possible estral cycling of these females, all were routinely examined and used only in diestrous. At the time of killing, the hypothalamus was dissected out using techniques employed previously by us and described elsewhere4,10. A sample of blood was obtained at this time, and an aliquot was analyzed fluorometrically to confirm the degree of success of adrenalectomy 2. The remaining blood sample was treated in a similar fashion as the tissue to check reproducibility of extraction of the radioactive steroid. This procedure has recently been described in detail by McEwen eta[. 7 and will, therefore, not be described here. The hypothalamic tissue was extracted with dichloromethane (DCM) overnight on a metabolic shaker. Generally, this procedure resulted in a 92-96'>,., extraction of DCM-soluble radioactive material. Thin-layer chromatography was used to identify labeled corticosterone in the DCM extracts of brain and blood. This step was necessary since recent data have indicated the conversion of corticosterone to l 1-dehydrocortisone,5 by brain tissue. The procedures for this analysis were essentially identical to those described previously v. After DCM extraction, the material was placed in a scintillation vial together with 15 ml of a toluene-based scintillation fluid and counted in a scintillation counter. At the completion of the experiment, the

GENETIC ANALYSISOF HYPO'IHALAMIC CORTICOSTERONE RETENTION

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d a t a were a n a l y z e d statistically by analyses o f variance, the S t u d e n t - N e w m a n - K e u l s multiple range test, and an e s t i m a t i o n o f T u k e y ' s W 1~. These analyses p e r m i t t e d us to arrive at a tentative h y p o t h e t i c a l genetic model for subsequent testing in e x p e r i m e n t 2. Experiment 2 Based on the findings o f e x p e r i m e n t 1, we m a d e the following crosses: ( l ) C X B K × C57BL/6By; ( 2 ) C X B J x C57BL/6By; ( 3 ) C X B K :4 B A L B / c B y ; (4) B6CF1 x C57BL/6By; (5) B 6 C F j x BALB/cBy. F e m a l e mice for these crosses, a n d the tissues derived f r o m these a n i m a l s were h a n d l e d in identical fashion to t h a t described in e x p e r i m e n t 1. The d a t a were a n a l y z e d statistically as described previously. In a d d i t i o n , b i m o d a l i t y o f the d a t a from the backcross mice B6CF1 x B A L B / c B y a n d B6CF1 x C57BL/6By was tested. It m u s t be e m p h a s i z e d t h a t because o f the e x t e n d e d time t a k e n to c o m p l e t e this w o r k , and in o r d e r to minimize seasonal variability at the time t h a t the various crosses a n d backcrosses were analyzed, several o f the a l r e a d y e x a m i n e d strains were analyzed again to observe possible deviations from the original analysis. T h e latter analyses, however, i n d i c a t e d no significant v a r i a t i o n s due to season.

TABLE I Mean level and statistically based magnitude rank order of DCM-extractabl¢ radioactivity ([aH]corticosterone) in the hypothalamus of female mice of the recombinant inbred strains, their progenitor sh'ains, Fj crosses and various backcrosses (means -L S.E.M.). Magnitude of level

Strains of mice

N

Radioactivity (counts/rain/rag wet tissue)

Low

BALB/cBy CXBI CXBK CXtlG CXBE B6CF: x BALB/cBy (low group)* CXBX × BALB/cBy

8 8 8 8 8 18 10

31.3 . 1.1 25.6 ± 2.4 32.7 --t. 1.5 34.5:5 1.8 45.3 :~ 6.5 28.6 ": 2.6 23.7 t 1.1

Intermediate

CB6F~ 8 B6CFI 8 B6CFI x BALB/cBy (intermediate group)* 11 B6CF1 x C57BL/6By (intermcdiategroup)* 15 CXBK x C57BL/6By 12

40.1 t. 6.9 59.7 -3:5.7 55.6 2:3.6 50.5 ~ 5.8 47.2 5_ 1.7

High

C57BL/6By CXBD CXBJ CXtlH B6CF~ x C57BL/6By (high group)* CXBJ × C57BL/6By

76.7 76.6 67.7 64.2 84.5 83.2

* For further clarification, see Fig. 1.

8 8 8 8 15 10

i: 5.2 ~ 12.6 -: 7.3 ± 8.1 -3 6.5 ~ 2.5

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I

II

III

o

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C57BL/6By

I I

I

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IIII

I

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O' (lntemediole)

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t

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I

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II

I I

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I

0

B6CF I x C57BL/6By

II il

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I I

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(High)

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cB6CF I + CB6F~

I II I

I I

I I II I Ill [

I

IJ I

0

il

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I

(LOW)

I llli

I I i

0

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(Intermediate) B6CF

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BALBIcBy I

I0

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25

40

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5,5 CPM

70

I

85

I

IO0

/ mg

Fig. I. Frequency distribution of the DCM-extractable radioactivity levels (countshnin,nlg) in the hypothalami of the parental strains, BALB/cBy and C57BL/6By, the reciprocal F~ hybrids, B6CFJ and CB6F], and the two backcrosses, B6CF1 ,: BALB/cBy and B6CFt C57BL/6By. Frequencies indicated are according to statistically separated groups, i.e. low, intermediate or high. (Mean ! S.E.M.)

RESULTS

The data on hypothalamic DCM-extractable radioactivity (counts/rain/rag wet tissue) are presented in Table | and Fig. 1. The results of various statistical analyses permitted grouping of the progenitor strains, reciprocal FI crosses and backcrosses and R! strains, into 3 major magnitude categories: low, intermediate and high (Table 1). Generally, of the two progenitor strains, BALB/cBy exhibited a low (31.3 5 1.1 counts/min/mg) while C57BL/6By exhibited a significantly (P ,: 0.01) higher radioactive count (76.7 :': 5.2). The 7 RI strains were distributed among the 3 categories, while the reciprocal F] hybrids exhibited an intermediate count. Of the 3 crosses, that of CXBK x BALB/cBy, both of low radioactive count, exhibited a low count, while that of CXBK (low radioactivity) i,, C57BL/6By (high radioactive count) exhibited an intermediate level, and that ofCXBJ ?~ C57BL/6By, both high, exhibited a high count. The F1 backcross to the progenitor strain BALB/cBy resulted in two distinct groups, one exhibiting low and the other exhibiting an intermediate radioactive count (Fig. 1). The FI backcross to the other parental strain, C57BL/6By, resulted in two distinct groups, one of intermediate, and the other of high radioactive count.

GENETIC ANALYSIS OF ItYPOTHAI,AMIC CORTICOS]'ERONE RETENTION

~1

DISCUSSION

Several aspects of the statistical grouping of strains and crosses, and the data obtained with the backcrosses (Table 1, Fig. I) permitted us to derive a genetic model of hypothalamic retention of [ZH]corticosterone on the following rationale: (1) parental strain BALB/cBy was low and C57BL/6 By was high in retention; (2) the reciprocal Ft hybrids exhibited an intermediate value indicating lack of dominance and no maternal influence; (3) cross of two low-retention strains, CXBK × BALB/cBy, resulted in offspring exhibiting low retention: (4) cross of two high-retention strains, CXBJ :~'. C57BL/6By resulted in offspring exhibiting high retention ; (5) the backcross of B6CFI by either parent resulted in two distinct groups of either low and intermediate, or intermediate and high (Fig. 1), respectively. Thus, based on these data, the assumption was made that this phenomenon is influenced genetically by at least one locus. Unfortunately, the strain distribution pattern of the RI lines did not permit further studies on linkage of this locus, since it did not match any of the available congenic lines to test such linkage. Since this is the tirst genetic study into the phenomenon of hypothalamic retention of [:~H]corticosterone, little can be said regarding the specific nature of this phenomenon. For example, we do not know if the genetic control is at the blood brain barrier, alters membrane permeability phenomena of specific neurones, or if il influences availability of specific soluble corticosterone-binding macromolecules". Alternatively, this locus may possibly influence one or more of these mechanisms. Further research is necessary to elaborate on this finding. Generally, the present data show that it is possible to ascribe to a single gene, a major effect of a complex neuroendocrine process. Additionally, this knowledge will permit experimentation into the specific control mechanisms of this phenomenon. and offers unique opportunities to elaborate on the genetic nature of the corticosterone-binding macromoleculesL ACKNOWLEDGEMENTS

Supported in part by NIH Research Grant HD-05860 from the National Institute of Child Health and Human Development and in part by funds from the Britton Foundation. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.

REFERENCES 1 Bat t.eV, D. W., Recombinant inbred strains, Transplantation, I1 (1971)404-407. 2 BUTTE, J. C., AND NOBLE, E. P., Simultaneous determination of plasma or whole blood cortisol and corticosterone, /fcta Endocr. (Kbh.), 61 (1969) 678-686. 3 ELEFTHERIOU, B. E., AND BAILEY, D. W., Genetic analysis of plasma corticosterone levels in t~o inbred strains of mice, J. Endocr., 55 (1972) 415-420.

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4 ELEETHERIOU, B. E., Effects of amygdaloid lesions on hypothalamic macromolecules. In B. E. ELF.FTHERIOU (Ed.), 7he Neurobiology of the Amygdala, Plenum Press, New York, 1972, pp. 793-813. 5 GROSSER, 13. l., 11 fl-Hydroxysteroid metabolism by mouse brain and g[ioma 26I, J. Neurochem., 13 (1966) 475-478. 6 MCEWEN, B. S., WEISS, J. M., AND SCHWARTZ, L.S., Selective retention of corticosterone by limbic structures in rat brain, Nature (Lond.), 220 0968) 911-912. 7 McEwEN, B. S., WEISS, J. M., AND SCHWARTZ, L. S., Uptake of corticosterone by rat brain and its concentration by certain limbic structures, Brain Research, 16 (1969) 227-241. 8 McEWEN, B. S., WEmS, J. M., AND SCHWARTZ, L. S., Retention of corticosterone by cell mlc[ei from brain regions of adrenalectomized rats, Brain Research, 17 (1970) 471-482. 9 McEWEN, B. S., WEISS, J. M., AND SCHWARTZ, L. S., Selective retention of corticosterone by limbic structures in rat brain, Endocrinology, 90 (1972) 217-226. 10 SIDMAN, R., ANGEVINE, J.B., TABER, E., AND PIERCE, S., Atlas of the Mouse Brain, Harvard Univ. Press, Cambridge, Mass., 1970. 11 WINER, B. J., Statistical Principles in Experimental Design, McGraw-Hill, New York, 1962.